Sybille Dezélée

The UL25 Protein of Pseudorabies Virus Associates with Capsids and Localizes to the Nucleus and to Microtubules

ABSTRACT The UL25 gene of pseudorabies virus (PrV) can encode a protein of about 57 kDa which is well conserved among herpesviruses. The UL25 protein of herpes simplex virus type 1 is a capsid constituent involved in virus penetration and capsid maturation. To identify and characterize the UL25 gene product of PrV, polyclonal mouse anti-UL25 antibodies were raised to a bacterially expressed fusion protein. In immunoblotting and immunoprecipitation assays of PrV-infected cell lysates, these anti-UL25 antisera specifically recognized a protein of the expected size with late expression kinetics. This 57-kDa product was also present in purified virions and was found to be associated with all types of capsids. Synthesis of a protein migrating at the same size point was directed from the eukaryotic expression plasmid pCG-UL25. To determine the subcellular localization of UL25, immunofluorescence studies with anti-UL25 antisera were performed on Nonidet P-40-extracted COS-7 cells infected with PrV or transfected with pCG-UL25. In PrV-infected cells, newly synthesized UL25 is directed mainly to distinct nuclear compartments, whereas UL25 expressed in the absence of other viral proteins is distributed more uniformly in the nucleus and colocalizes also with microtubules. To study the fate of UL25 at very early stages of infection, immunofluorescence experiments were performed on invading PrV particles in the presence or absence of drugs that specifically depolymerize components of the cytoskeleton. We found that the incoming nucleocapsids colocalize with microtubules during their transport to the nucleus and that UL25 remains associated with nucleocapsids during this transport.

Control of Sigma Virus Multiplication by the ref(2)P Gene of Drosophila melanogaster : An in Vivo Study of the PB1 Domain of Ref(2)P

Ref(2)P has been described as one of the Drosophila proteins that interacts with the sigma virus cycle. We generated alleles to identify critical residues involved in the restrictive (inhibiting viral multiplication) or permissive (allowing viral multiplication) character of Ref(2)P. We demonstrate that permissive alleles increase the ability of the sigma virus to infect Drosophila when compared to null alleles and we confirm that restrictive alleles decrease this capacity. Moreover, we have created alleles unfunctional in viral cycling while functional for Ref(2)P fly functions. This type of allele had never been observed before and shows that fly- and virus-related activities of Ref(2)P are separable. The viral status of Ref(2)P variants is determined by the amino-terminal PB1 domain polymorphism. In addition, an isolated PB1 domain mimics virus-related functions even if it is similar to a loss of function toward fly-related activities. The evolutionary tree of the Ref(2)P PB1 domain that we could build on the basis of the natural allele sequences is in agreement with an evolution of PB1 domain due to successive transient selection waves.

Further characterization of yeast RNA polymerases. Effect of subunits removal.

Two forms of yeast RNA polymerase A are resolved by phosphocellulose chromatography. One of these, called RNA polymerase A, is lacking two polypeptide chains of 48,000 and 37,000 daltons. The properties of the two enzymes are compared in the present paper. RNA polymerase A transcribes d(A-T)n with a similar efficiency as the complete enzyme, but it is comparatively much less active with native DNA. The two enzymes can also be differentiated on the basis of their ionic strength and divalent cation requirements. RNA polymerase A has a particularly low activity at high salt and low Mg2+ concentrations. Thermal inactivation curves of the two enzymes are different when residual activity is assayed with native DNA. In contrast with d(A-T)n as template the apparent inactivation curves of the two enzymes are identical. The data suggest that the two dissociable polypeptide chains play an important role in transcription. The template specificity of yeast RNA polymerase B was further investigated using SV40 DNA-FI as template. RNA polymerase B is able to retain [3H]SV40 DNA-FI on nitrocellulose filters but the enzyme-DNA complex is very unstable. The observation that RNA polymerase B can transcribe to some extent a supercoiled DNA but not a linear double stranded template supports the hypothesis that the enzyme needs some unpaired DNA structure to initiate transcription.

Vesicular Stomatitis Virus Growth in Drosophila melanogaster Cells. II. Modifications of Viral Protein Phosphorylation

The phosphoproteins of vesicular stomatitis virus released from infected Drosophila melanogaster cells were examined. The membrane (M) protein was more phosphorylated than after multiplication in chicken embryo cells, even in Drosophila cell cytoplasm before its association with cellular membranes. Analysis of phosphopeptides generated after partial proteolysis and of phosphoamino acids obtained after complete acid hydrolysis showed that M phosphorylation was quantitatively and qualitatively changed, while NS protein phosphorylation was only slightly modified.

Sigma virus: growth in Drosophila melanogaster cell culture; purification; protein composition and localization

Summary The growth cycle of sigma virus in Drosophila melanogaster cells was studied: optimal virus production was reached about 40 h after infection; virus release declined thereafter and then remained approximately constant (carrier state). In the presence of DEAE-dextran during virus adsorption, more cells became infected and sigma virus production was enhanced. Sigma virus was partially purified by a gentle procedure. Five presumptive virus-specific proteins with molecular weights 210K, 68K, 57K, 44K and 25K were observed. The p68 polypeptide was glycosylated and formed the spikes of the virion particles. The nucleocapsid contained a single major protein, p44, and one or two minor proteins (p210 and probably p57); another protein, p25, was more loosely associated with the nucleocapsid. None of these proteins was found to be phosphorylated.

Yeast RNA Polymerases

INTRODUCTION The presence of multiple forms of RNA polymerases has been recognized in all eukaryotic cells, including plant, insect, fungi and yeast cells (Chambon 1975). A basic, generally accepted assumption is that these related enzymes are specifically synthesizing the various classes of RNA (Zylber and Penman 1971; Reeder and Roeder 1972; Weinmann and Roeder 1974; Chambon 1975). In animal cells, the α -amanitin-sensitive class B (or II) RNA polymerase is believed to synthesize precursor messenger RNA; the moderately sensitive class C (or III) enzyme is responsible for the synthesis of 5S and tRNA; whereas class A (or I) enzyme, which is insensitive to high concentrations of the toxic peptide, probably makes ribosomal RNA. Although it is still premature to extend this conclusion to lower eukaryotic cells, such as yeast cells, the striking similarity between enzymes isolated from various types of cells strongly supports this general hypothesis. One important problem, then, is to understand what determines, on a structural basis, the different specificities of the three classes of RNA polymerase. Additional complexity arises from the presence of subclasses of RNA polymerases. For instance, RNA polymerase B can be resolved into two or three forms of enzyme, B0 (II 0 ), BI (II A ) and BII (II B ), which differ only in the molecular weight of their largest subunit (Chambon 1975; Link and Richter 1975; Schwartz and Roeder 1975). Two forms of RNA polymerase C can also be separated (Schwartz et al. 1974). The in vivo significance of this multiplicity is unclear. Therefore the origin and possible...

Restricted Expression of Viral Glycoprotein in Vesicular Stomatitis Virus-infected Drosophila melanogaster Cells

Vesicular stomatitis virus (VSV) establishes a non-cytopathic persistent infection in Drosophila melanogaster cells. The synthesis of the viral glycoprotein G was specifically inhibited during a post-transcriptional step, whereas the synthesis and turnover of its mRNA were not modified compared with the other viral mRNAs. Another viral glycoprotein, migrating slightly faster than G protein on an SDS-polyacrylamide gel, was detected in infected Drosophila cells. This protein showed most of the characteristics of the intracellular Gs protein found in infected vertebrate cells. The amounts of G protein integrated into mature virions and of soluble Gs protein secreted into the culture medium were reduced greatly during VSV infection in Drosophila cells.